The present application relates to, but is not limited to, selective capture and release of analytes. For example, the present application relates to minimally invasive extraction, purification and concentration (PC) of analytes.
A need exists for techniques to selectively capture and release analytes with minimal harm to the analytes. For example, such techniques are applicable to extraction of analytes for biochemical analysis. Other applications include detection of harmful components in pharmaceuticals or food, extraction of harmful environmental agents, selective release of drugs at a target location in the body, and the like.
As an example, there is a desire to develop highly integrated biological analysis devices that can be used to perform general biochemical analysis. One component in these devices is sample preparation, which involves extraction and PC of applicable analytes.
Some techniques have employed solid-phase (SP) gels for retention of target molecules. A common shortcoming of SP devices is that their capture mechanisms are often indiscriminate with respect to the target analyte. For example, hydrophobic and ion-exchange SP device are limited because they extract impure compounds with similar physical or chemical properties as the target. With applications in drug delivery or chemical assays, where specific molecules need to be released, introducing impurities can be problematic. In addition, elution of molecules using harsh pH or solvent gradients is common in SP devices. For certain biomedical applications, these elution schemes can present potential health hazards. Furthermore, it is desirable to selectively release the captured molecules for applications in which their use is location-specific.
Biotechnology research, such as proteomics and genomics, utilizes biological mass spectrometry, which is label-free and offers increased resolution detection. In particular, matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is useful because it permits relatively simple data interpretation, good detection limits and parallel processing. MALDI-MS is based on a soft ionization technique in which analytes are cocrystallized with an energy-absorbing matrix material on the surface of a substrate (called a MALDI analysis plate). Notwithstanding its broad utility, the overall quality and efficacy of quantifiable MALDI-MS generally depends on the purity of the introduced sample. Techniques involving sample preparation, such as analyte extraction, have increasingly been employed to condition biological samples prior to MALDI-MS analysis. This can entail the separation, purification and concentration of analytes preceding quantitative analysis. For example, analyte extraction can be used to retrieve and isolate a rare analyte from a complex mixture of undesirable constituents such as salts, particulates, solvents or physiological tissue so as to enrich and enhance the analyte's MALDI-MS detection.
Solid-phase extraction (SPE) as a sample preparation procedure prior to MALDI-MS that can be used to provide pure and concentrated samples to enable increased sensitivity analysis. During SPE, an analyte of interest within a fluid phase is exposed to a solid phase (e.g., microbeads coated with a thin layer of a functional material). The analyte interacts through surface chemistry with the coating and therefore is retained by the solid phase. This allows impurities and non-target compounds remaining in the liquid phase to be removed by rinsing. Next, a reagent (such as an organic solvent) is generally used to disrupt the interaction between the solid phase and the analyte, thereby eluting the analyte for further analysis. Other sample preparation techniques include electrokinetic sample stacking, liquid-liquid extraction, and dialysis. The off-line nature of MALDI-MS makes it suitable for coupling to off-line SPE approaches, which facilitate high-throughput processing designs with small dead volumes.
In some SPE protocols, one challenge is to effectively concentrate and purify minute quantities of analytes, while minimizing absorptive losses and maximizing recovery in as compact an elution volume as possible. Microfluidic technology has been utilized to attempt to overcome this obstacle. Miniaturization helps facilitate the handling of limited sample quantities, the reduction of dead volumes, an increase the effective surface-to-volume ratio to promote efficient chemical reactions, and integration. Also, microfabrication allows for massive parallelization of sample processing, while being amenable to MALDI-MS which can lower analysis costs. Existing microfluidic SPE devices utilize physisorption capture of the target analyte by gels or membranes. For example, some techniques use a commercial reversed-phase gel (Poros) on some microfabricated silicon chips for sample enrichment of alcohol dehydrogenase. The proteins are eluted by addition of a polar solvent (e.g., acetonitrile), which changes the surface polarity of the support to release the bound analyte. Ion-exchange supports, such as some methacrylate based gels, depend on adjustment of charged molecules on the retention media to interact with analytes. Strong pH reagents can be introduced to subsequently release the molecules of interest. Alternatively, other techniques use a packed 2.5 mm column of C18 microbeads for the reverse-phased preconcentration of ephedrine on a poly (vinylpyrrolidone) chip which is then eluted using an acetonitrile-borate buffer solution.
Existing microfluidic SPE devices, however, remain inadequate to address the current demands in MALDI-MS analysis, which increasingly requires processing of complex biological or chemical samples, such as blood, serum, or tissue mass. A given analyte should be detectable amongst cellular debris, non-specific molecules, and salts within such samples. Standard functional chemistries for solid-phase purification often lack selectivity to target analytes since impurities usually exhibit similar physical properties (e.g., hydrophobicity or ionic charge) which allow their simultaneous retention. For unambiguous, sensitive detection of biomolecules by MALDI-MS, it is useful that the analyte extraction be specific, e.g., the analyte and no impurities are retained by the solid phase. Moreover, recovery of biomolecules using traditional techniques generally requires an adjustment in pH or application of a solvent gradient. This can compromise the integrity of sensitive compounds (which can already be in rare supply) and can further complicate the protocol by requiring the handling of potentially harsh reagents.
Biosensors are used for the detection and analysis of biomolecules that are disease relevant biomarkers such as genes, proteins, and peptides. They can include of a molecular recognition component and a transducer converting the binding event into a measurable physical signal. An important class of biosensors includes affinity biosensors, which rely on highly selective affinity receptors recognizing target biomolecules. Traditionally used affinity receptors include antibodies and enzymes, which are known to have limitations such as instability, poor regeneration, and physiologically-dependent production. These limitations can be addressed by biosensors that employ alternative, synthetically generated affinity receptors, in particular aptamers.
Aptamers include oligonucleotides that recognize target molecules specifically by highly selective affinity interaction; they are isolated through a synthetic procedure called systematic evolution of ligands by exponential enrichment (SELEX), whereby very large populations of random sequence oligomers (DNA or RNA libraries) are screened against the target molecule in an iterative procedure. Aptamers have been developed to target a variety of biomolecules (e.g., small molecules, peptides, and proteins) in diverse applications, such as target validation, drug discovery, and in particular, diagnostics and therapy. The intense attention received by aptamers can be attributed not only to their high specificity, but also to characteristics that are lacking in more established affinity receptors such as enzymes, lectins, and antibodies. These include enhanced stability at room temperature, and more easily modified terminal ends, as compared to their conventional affinity receptor counterparts (e.g., antibodies and enzymes), so as to facilitate attachment to stationary surfaces. Moreover, aptamer-target binding is generally reversible under changes in environmental parameters such as pH and temperature. Thus, aptasensors can be regenerated via such experimental stimuli, which can also be exploited to allow controlled release and recovery of target biomolecules.
Microelectromechanical systems (MEMS) have been applied to biosensing, leading to minimized sample consumption, improved robustness and reliability, reduced costs, and the possibility of parallelized, high-throughput operation. In particular, microfluidic devices have been used for affinity biosensing, such as microcantilever immunosensors for myoglobin and nanoparticle-antibody conjugated array sensors for detecting food-born Escherichia coli. Microcantiliever aptasensors have been used for specific detection of Thermus aquaticus DNA polymerase. Biomolecules are detected after binding by monitoring surface stress induced deflection of the cantilever by an interrogating light source. Alternatively, love-wave microfluidic aptasensors have been used to detect multifunctional serine protease thrombin and Rev peptide, fabricated from polymethylmethacrylate on top of a quartz substrate. Nanostructures such as single-walled carbon nanotubes have been functionalized with aptamers to detect thrombin. The selectivity of the thrombin aptamer has been tested against elastase to which the conductance of the SWNT-FET showed no change.
Aptasensing of arginine vasopressin (AVP) for the diagnosis and therapy of septic shock (induced by severe infection) and congestive heart failure, conditions that restrict the cardiovascular system's ability to provide adequate perfusion in order to maintain organ functionality is a clinical application of aptasensors. Both disorders are indicated by elevated levels of AVP, a cyclic polypeptide neurohormone that is synthesized in the hypothalamus and promotes vasoconstriction. Specifically, physiological concentrations of AVP in plasma markedly increases up to tenfold that of average levels (5-10 pM) in order to maintain arterial pressure and hence, blood perfusion. As shock progresses however, the initial abundance of AVP in plasma decreases. Thus, the ability to monitor and control AVP over time can reveal the homeostatic status of the patient, and could potentially provide therapeutic solutions for septic shock and congestive heart failure. Platforms for vasopressin include immunoradiometric assays (IRA) and enzyme-linked immunosorbent assays (ELISA). The use of these assays is often hindered by several limitations: time-consuming and complicated radio and fluorescent labeling protocols; excessive use of sample and auxiliary reagents; and limited long-term stability and shelf-life. Moreover, prolonged incubation times can result in slow diagnostic turnaround (3-11 days), which renders these techniques rather ineffective for therapeutic management of AVP.